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WO2021172890A1 - Anticorps monoclonal se liant spécifiquement à tim-3 et ses utilisations - Google Patents

Anticorps monoclonal se liant spécifiquement à tim-3 et ses utilisations Download PDF

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WO2021172890A1
WO2021172890A1 PCT/KR2021/002369 KR2021002369W WO2021172890A1 WO 2021172890 A1 WO2021172890 A1 WO 2021172890A1 KR 2021002369 W KR2021002369 W KR 2021002369W WO 2021172890 A1 WO2021172890 A1 WO 2021172890A1
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tim
antibody
antigen
seq
variable region
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PCT/KR2021/002369
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English (en)
Korean (ko)
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박은정
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국립암센터
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Priority to CN202180016902.2A priority Critical patent/CN115151571A/zh
Priority to US17/904,922 priority patent/US20230087570A1/en
Priority to EP21761657.2A priority patent/EP4112643A4/fr
Publication of WO2021172890A1 publication Critical patent/WO2021172890A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Definitions

  • the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3 and uses thereof.
  • T cell immunoglobulin and mucin domain-3 is a transmembrane glycoprotein composed of an immunoglobulin variable domain, a mucin domain, and a cytoplasmic tail with a tyrosine phosphorylation motif.
  • TIM-3 was proposed as a T helper type 1 (Th1)-specific protein selectively expressed in terminally differentiated Th1 cells, but as a result of further studies, TIM-3 is a Th17 cell, regulatory T (Treg) cell, and even non-T cells such as dendritic cells (DC), natural killer (NK) cells, monocytes and macrophages. This suggests that TIM-3 has various immune functions depending on specific cell types and cell states.
  • TIM-3 seems to play both positive and negative roles depending on the situation.
  • TIM-3 can attenuate the response of CD4+ and CD8+ T cells and inhibit T cell activation to induce peripheral tolerance.
  • TIM-3 may contribute to the elimination of pathological stimuli by participating in the activation of various innate immune cells, including quiescent macrophages. Recent studies have shown that TIM-3 promotes short term effector T cells during viral infection. It has also been reported that dysregulation of TIM-3 expression responds excessively or inappropriately in immune cells.
  • TIM-3 in many diseases, including autoimmune diseases, chronic infections and ischemia. Alterations and dysregulation of TIM-3 expression have been associated with the onset and severity of pathological conditions in patients with multiple sclerosis (MS) and patients infected with human immunodeficiency virus (HIV) or hepatitis virus, particularly IFN- ⁇ in MS patients. and high levels of expression of TNF- ⁇ as well as distortion of Th1-induced Th2 responses in allergic diseases. Moreover, blockade of TIM-3 has been shown to affect the pathological severity of experimental allergic encephalomyelitis (EAE) and the pathogenesis of diabetes in obese diabetic (NOD) mice.
  • EAE experimental allergic encephalomyelitis
  • NOD obese diabetic
  • TIM-3 signaling restored proliferation and increased cytokine production in HIV-specific T cells.
  • negative regulation of Th1 or Th17 immunity by TIM-3 is associated with T cell dysfunction or depletion of T cells, distortion of Th2 responses to Th1 signaling, and proinflammatory states of Th1 responses. It has the potential to cause the same pathological condition.
  • TIM-3 is also receiving attention as a possible target for immune modulation of cancer.
  • many studies have been conducted on immune checkpoint molecules and immune surveillance in the context of cancer growth and eradication, which has led to the development of numerous therapeutic strategies, including therapeutic antibodies targeting immune checkpoint molecules.
  • Studies have suggested that TIM-3 expression is associated with several cancers and that TIM-3 plays a role in regulating tumor growth.
  • TIM-3 is highly expressed on CD4+ and CD8+ tumor-infiltrating T cells from patients with non-small cell lung cancer, and TIM-3 expression on CD4+ T cells is associated with poor clinicopathological parameters such as nodular metastasis and advanced cancer. It was found to be characteristically expressed in tumor cells in patients with renal and hepatocellular carcinoma.
  • Korean Patent Registration No. 10-2146319 relates to a bispecific antibody specific for PD-1 and TIM-3.
  • Bispecific antibodies comprising a -binding site, in particular bispecific antibodies that bind to TIM3 with a lower binding affinity compared to binding to PD1 are provided.
  • the present inventors have made and selected the monoclonal antibody of the present invention and completed the present invention as a result of earnest efforts to provide an antibody that specifically binds to TIM-3 with high affinity.
  • the present invention provides a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; and
  • a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6;
  • the antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; And a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; may include.
  • the heavy chain variable region and the light chain variable region may be connected by a linker consisting of the amino acid sequence of SEQ ID NO: 9.
  • the antibody may be a humanized antibody or a human antibody.
  • the present invention also provides a nucleic acid molecule encoding a heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof or a nucleic acid molecule encoding a light chain variable region.
  • the nucleic acid molecule may consist of the nucleotide sequence of SEQ ID NO: 16 or the nucleotide sequence of SEQ ID NO: 17.
  • the present invention can provide a nucleic acid molecule encoding the heavy chain variable region, a nucleic acid molecule encoding the light chain variable region, or a recombinant vector containing all of the nucleic acid molecule.
  • the present invention can provide a host cell comprising the recombinant vector.
  • the present invention may provide a method for producing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3, comprising the step of culturing the host cell.
  • the present invention may provide a kit for measuring TIM-3 expression level comprising the monoclonal antibody or antigen-binding fragment thereof.
  • the present invention comprises the steps of: i) contacting the monoclonal antibody of claim 1 or an antigen-binding fragment thereof with a biological sample isolated from a subject;
  • the biological sample may be any one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, tissue and cells.
  • the monoclonal antibody hTIM-3_NCC1 of the present invention specifically recognizes human and mouse cells expressing TIM-3, and various fields such as diagnosis of diseases mediated by TIM-3 expressing cells as well as prevention or treatment thereof can be useful for
  • FIG. 1 shows the ELISA results of antibody candidates specific for TIM-3. They correspond to scFv-TIM3#1 to scFv-TIM3#12 in order from left.
  • hTIM-3_NCC1 antibody reacts and specifically binds to human TIM-3 in human PBMCs better than a commercially available anti-human TIM-3 antibody (BV421 ⁇ -Human TIM-3, BD#565553). indicates to do
  • hTIM-3_NCC1 antibody can also bind to mouse TIM-3.
  • hIgG Fc refers to APC-hIgG Fc
  • ⁇ -hTIM-3 refers to the antibody hTIM-3_NCC1 of the present invention.
  • Rat Isotype-PE (eBioscience, cat#12-4321) is an isotype antibody of ⁇ -mTIM-3-PE antibody (eBioscience, cat#12-5870), and APC-hIgG Fc (Biolegend, cat#409305) denotes a secondary antibody control against ⁇ -hTIM-3-scFv-hIgG (hTIM-3_NCC1).
  • Antigen of the present invention refers to a molecule or part of a molecule that can be used in an animal to produce an antibody capable of binding by an antibody and also capable of binding to an epitope of an antigen.
  • An antigen may have more than one epitope.
  • antibody refers to a specific target or antigen, such as carbohydrate, polynucleotide, lipid, polypeptide, etc. It is an immunoglobulin molecule that can "Antibody” means monoclonal antibody; polyclonal antibody; "Antigen binding fragments” of antibodies that retain the ability to specifically bind to a particular antigen (eg, TIM-3), such as Fab, Fab', F(ab')2, Fd, Fv, Fc etc; an isolated complementarity determining region (CDR); bispecific antibodies; heteroconjugate antibodies, mutants thereof; a fusion protein having an antibody, or antigen-binding fragment thereof (eg, a domain antibody); single chain (ScFv) and single domain antibodies (eg, shark and camelid antibodies); maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv; humanized antibodies; chimeric antibodies; and
  • CDR complementarity determining region
  • Antibodies or polypeptides that "specifically bind" to a particular target or antigen are terms well understood in the art, and methods of determining such specific binding are also widely known in the art. is known.
  • a particular molecule is said to exhibit "specific binding” when it reacts or associates with a particular cell or substance more frequently, more rapidly, with greater persistence and/or with greater affinity than it does with another cell or substance. It is considered A particular antibody “specifically binds” to a particular target or antigen if it binds with greater affinity, binding activity, more rapidly and/or with greater persistence than it does with other substances.
  • a "vector" of the present invention includes a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked.
  • a "plasmid” which refers to a circular double-stranded DNA loop to which additional DNA segments can be bound.
  • a viral vector in which additional DNA segments can be bound to the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having replication of bacterial origin and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby being replicated along the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors (or simply, “expression vectors”).
  • expression vectors useful in recombinant DNA technology often exist in the form of plasmids.
  • plasmid and vector can be used interchangeably because plasmid is the most commonly used form of vector.
  • the present invention provides other forms of expression vector with equivalent function, e.g., viral vector (e.g., replication deficient) retroviruses, adenoviruses and adeno-associated viruses).
  • “Host cell” in the present invention is used to mean a cell that is transformed or is transformed with a nucleic acid sequence and then is capable of expressing a selected gene of interest.
  • the term includes progeny of a parent cell, whether morphologically or genetically identical to the original parent, as long as the selected gene is present.
  • the present inventors have completed the present invention as a result of earnest efforts to provide an antibody that can be usefully used for TIM-3 monitoring or disease diagnosis through specific and selective strong binding to TIM-3.
  • the antibody hTIM-3_NCC1 of the present invention can specifically and selectively bind not only human TIM-3 but also mouse TIM-3, it is very suitable for monitoring TIM-3 expression levels. Therefore, when using the antibody hTIM-3_NCC1 of the present invention, since the expression level of TIM-3 can be measured very accurately, the field for checking the TIM-3 expression level, for example, diagnosis of diseases such as cancer, multiple sclerosis or brain disease Alternatively, it can be widely used in the field of predicting the prognosis or the field of preclinical research using mice.
  • the present invention provides a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; and
  • a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6;
  • the "antigen-binding fragment thereof” refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv or single-chain antibody molecules.
  • Fab has a structure having variable regions of light and heavy chains, constant regions of light chain and the first constant region (CH1) of heavy chain, and has one antigen-binding site.
  • F (ab') differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F (ab')2 is generated when a cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • Fv refers to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region.
  • Such an antibody fragment can be obtained using a proteolytic enzyme, for example, by restriction digestion of the entire antibody with papain to obtain Fab, and digestion with pepsin to obtain a F (ab')2 fragment, preferably can be produced through genetic recombination technology.
  • the antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; And a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; may include.
  • the heavy chain variable region and the light chain variable region may be connected by a linker consisting of the amino acid sequence of SEQ ID NO: 9.
  • the antibody may be a humanized antibody or a human antibody.
  • the humanized antibody is engineered to contain an immunoglobulin domain that is more and more human-like, which contains only the complementarity determining regions of an animal-derived antibody. This is achieved by carefully examining the sequences of the hypervariable loops of the variable regions of monoclonal antibodies and adapting these sequences to the structure of the human antibody chain.
  • a fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including the CDRs, arises from human genes.
  • the present invention also provides a nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof; Alternatively, a nucleic acid molecule encoding a light chain variable region may be provided.
  • nucleic acid molecule has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include natural nucleotides as well as analogs with modified sugar or base sites. also includes The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
  • the nucleic acid molecule of the present invention is to be construed to include a nucleotide sequence exhibiting substantial identity to the above-described nucleotide sequence.
  • the substantial identity is at least 80 when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. % homology, in one specific example at least 90% homology, in another specific example at least 95% homology.
  • the nucleic acid molecule encoding the heavy chain variable region may consist of the nucleotide sequence of SEQ ID NO: 16
  • the nucleic acid molecule encoding the light chain variable region may consist of the nucleotide sequence of SEQ ID NO: 17 it could be
  • the present invention can also provide a nucleic acid molecule encoding the heavy chain variable region, a nucleic acid molecule encoding the light chain variable region, or a recombinant vector comprising all of the nucleic acid molecule.
  • the recombinant vector system of the present invention can be constructed through various methods known in the art.
  • Vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • the present invention can also provide a host cell comprising the recombinant vector.
  • the host cell is a cell transformed with the recombinant vector of the present invention.
  • a host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art and any host cell can be used, for example, Escherichia coli, Bacillus subtilis and Bacillus thuringensis, such as Bacillus spp., Streptomyces, Pseudomonas (eg, Pseudomonas putida), Proteus mirabilis ( Proteus mirabilis) or Staphylococcus (eg, Staphylococcus carnosus), such as prokaryotic host cells.
  • the present invention may also provide a method for producing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3, comprising culturing the host cell.
  • the culture of the host cells for the production of the antibody or antigen-binding fragment thereof may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain.
  • Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch and continuous culture methods according to the culture method.
  • the medium used for culture should suitably satisfy the requirements of a particular strain.
  • the present invention may also provide a kit for measuring TIM-3 expression level comprising a monoclonal antibody or antigen-binding fragment thereof.
  • the expression level is measured by a method capable of measuring antigen-antibody binding, and the method may include any immunological analysis method.
  • immunological analysis method are known in the art, for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion assay, immunofluorescence assay, immunoblot, Oucreroni immunodiffusion assay, rocket immunoelectrophoresis, tissue immunostaining, immunization It may be a precipitation assay, a complement fixation assay, an immunochromatographic assay, FACS, or a protein chip.
  • Tools or reagents used in immunological assays may include suitable carriers or supports, labels capable of generating a detectable signal, solubilizing agents, detergents, and stabilizers, and the like.
  • suitable carriers include, but are not limited to, when the labeling material is an enzyme, a substrate capable of measuring enzymatic activity, an appropriate buffer solution, a chromogenic enzyme or a fluorescent substance-labeled secondary antibody, a chromogenic substrate, and reaction stop and the like.
  • the label includes any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Suitable labels include, but are not limited to, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g., For example, 3 H, 125 I, 35 S, 14 C or 32 P), enzymes (eg, mustard radish peroxidase, alkaline phosphatase, luciferase and enzyme-linked immunosorbent assay (ELISA)) commonly used) and colorimetric labels such as colloidal gold or colored glass or plastic (eg polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.
  • the antibody included in the kit of the present invention may be immobilized on a suitable carrier or support using various methods as disclosed in the literature, and examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester. , polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterin, gabbro, filter paper, ion exchange resin, plastic film, plastic tubes, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, metal, glass, glass beads, or magnetic particles and the like.
  • suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester. , polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose
  • solid substrates include cell culture plates, microplates, microarrays, ELISA plates, tubes and polymeric membranes.
  • the support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).
  • the present invention also provides: i) contacting the monoclonal antibody or antigen-binding fragment thereof with a biological sample isolated from a subject;
  • the subject of step i) may be a mammal including a human.
  • the biological sample may be any one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, tissue and cells.
  • TIM-3 specific antibodies (hTIM-3_NCC1, scFv-TIM3 #4) were selected through screening and ELISA reaction of a human scFv (naive) library using human TIM-3 antigen.
  • TIM-3-His tag (ACRObiosystems Inc.) was diluted in PBS at 2 ⁇ g/ml and dispensed on a plate by 100 ⁇ l per well. After the antigen was shaken off, 100 ⁇ l of 5% skim milk in PBS solution was added per well, reacted at room temperature for 4 hours, and then washed with PBS 4 times or more. ScFv binding to TIM-3-His tag by bio-panning an antibody library of scFv expressed on the surface of M13 phage in a 96-well plate to which Tim-3 protein antigen is attached. was collected.
  • TIM-3 specific antibody candidates obtained through the screening of the human ScFv (naive) library were diluted in 1% skim milk in TBST, respectively, and treated in each well, After reacting at room temperature for 1 hour, it was washed 3 times with 1X PBST. Secondary antibody (anti-human IgG HRP, 1:5000) was treated and reacted by shaking at room temperature for 1 hour, washed 6 times with 1X PBST, and then treated with 50 ml of TMB solution for color development at room temperature for 30 minutes. reacted for a minute. Finally, 100 ml of 1N H 2 SO 4 stop solution was treated for 10 minutes to terminate the reaction, and then absorbance was measured at 450 nm.
  • ScFV-TIM3#2, ScFV-TIM3#4, ScFV-TIM3#7, ScFV-TIM3#8 and ScFV-TIM3#12 antibodies were identified among 12 candidates, and the ScFV-TIM3#4 (a-hTIM-3-scFv-hIgG), which showed the strongest signal, was selected.
  • the selected ScFV-TIM3#4 clone was named hTIM-3_NCC1, and its base and amino acid sequences were analyzed. According to the sequence analysis results, sequence information on the antibody CDR region, that is, the heavy chain variable region and the light chain variable region was confirmed (Table 1, FIG. 2).
  • TIM-3-His tag ACRObiosystems Inc.
  • PBS PBS
  • 100 ⁇ l per well was dispensed on the plate.
  • 100 ⁇ l of 5% skim milk in PBS solution was added per well, reacted at room temperature for 4 hours, and then washed 4 times or more with PBS.
  • hTIM-3_NCC1 was diluted in 1% skim milk in TBST and treated in each well, followed by reaction at room temperature for 1 hour, and then washed 3 times with 1 X PBST.
  • Antigen TIM-3-His-tag 200 ng/well Primary Ab Anti-TIM-3-hscFv-hIgG1 Secondary Ab-HRP 1:3,000 TMB substrate incubation 10 min Measurement filter 450nm plate reader Infinite F50
  • hTIM-3_NCC1 specifically binds to TIM-3 as shown in FIG. 3 .
  • the amount of antigen coated on the immunoplate was about 0.2 ⁇ g (200ng/well) per well.
  • the antibody hTIM-3_NCC1 specifically binds to cells expressing TIM-3.
  • Lenti particles encoding TIM3 cDNA prepared using Lenti-Human TIM-3-GFP plasmid DNA (Origene, cat#RC209440L4) and Lenti-control particles-GFP (Origene, cat#PS100093V) were used in mice.
  • Human TIM-3 was overexpressed in NIH/3T3 cells as follows: 1 Lenti-control particles-GFP as a control group 5 ⁇ 10 4 NIH3T3 cells Lenti particles of 10 MOI were treated with polybrene Only the transduced cells were selectively selected using puromycin (NIH3T3-GFP).
  • Lenti-Human TIM-3-GFP plasmid DNA was transfected with packing DNA into 293FT cells, and a medium containing lenti-Human TIM-3-GFP particles obtained 3 days after transfection was added to NIH/3T3 with polybrene. handle Only cells overexpressing human TIM-3 were selectively selected using puromycin (NIH3T3-GFP-Human TIM-3).
  • PBMC Peripheral Blood Mononuclear Cells were obtained by Ficoll-Paque density gradient centrifugation using Ficoll-Paque (GE healthcare, cat#17-5442-02). , PBMC) was isolated separately. 1 ⁇ 10 6 isolated PBMCs, 0.5 ⁇ g of selected antibody, 1 ⁇ g of PE-Cy7-conjugated anti-CD3 antibody (BD, cat#557851, USA), 1 ⁇ g of FITC-conjugated anti-CD11b antibody (ebioscience, cat #11-0112, USA) 0.25 ⁇ g was reacted for 30 minutes, the surface was stained with a secondary antibody secondary antibody (PE-Human IgG Fc), and then measured by flow cytometry.
  • PE-Human IgG Fc secondary antibody secondary antibody
  • BV421-conjugated anti-TIM-3 antibody (BV421-Human TIM-3; BD, cat#565553, USA) was used as a positive control.
  • Levels of TIM-3 were measured in CD3 high CD11b - , CD3 high CD11b mid , CD3 - CD11b mid and CD3 - CD11b high cells.
  • hTIM-3_NCC1 reacts by recognizing TIM-3 expressed by cell groups expressing CD3 and CD11b of human PBMC, and has superior reactivity than a commercially available antibody used as a positive control. could check
  • hTIM-3_NCC1 FACS analysis was performed to determine whether the selected antibody, hTIM-3_NCC1, specifically binds to mouse TIM-3 as well as human TIM-3.
  • the antibody hTIM-3_NCC1 0.5 to mouse primary glial cells ⁇ g was reacted for 30 minutes, and then stained with a secondary antibody (APC-human IgG Fc; Biolegend, cat#409305).
  • mouse CD11b + cells (3 ⁇ 10 5 cells) overexpressing mouse TIM-3 were separated using a BV421-conjugated anti-CD11b antibody (a-BV421-conjugated a-mCD11b; BD, cat#562605).
  • a-BV421-conjugated anti-CD11b antibody a-BV421-conjugated a-mCD11b
  • BD cat#562605
  • hTIM-3_NCC1 also responds to TIM-3 expressed in mouse primary glial cells.
  • mouse hTIM-3_NCC1 antibody was also reactive with TIM-3 of the mouse cells.

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Abstract

La présente invention concerne un anticorps monoclonal ou un fragment de liaison à l'antigène de celui-ci qui se lie spécifiquement à TIM-3, et ses utilisations. L'anticorps monoclonal hTIM-3_NCC1 de la présente invention reconnaît spécifiquement des cellules humaines et murines exprimant TIM-3, et peut avantageusement être utilisé dans divers domaines, tels que le diagnostic d'une maladie médiée par des cellules exprimant TIM-3 et la prévention ou le traitement de celles-ci.
PCT/KR2021/002369 2020-02-25 2021-02-25 Anticorps monoclonal se liant spécifiquement à tim-3 et ses utilisations WO2021172890A1 (fr)

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CN114751985A (zh) * 2022-06-07 2022-07-15 日照市疾病预防控制中心 Tim-3抗体、制备方法及其应用

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KR20170075778A (ko) * 2014-10-27 2017-07-03 에이전시 포 사이언스, 테크놀로지 앤드 리서치 항-tim-3 항체
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